The present invention relates to transcellular transport of calcium, and in particular to compositions encoding calcium-transport proteins.
Calcium is a major component of the mineral phase of bone, and in ionic form plays an important role in cellular signal transduction. In particular, a signaling ligand (the xe2x80x9cfirst messengerxe2x80x9d) such as a hormone may exert an effect on a cell to which it binds by causing a short-lived increase or decrease in the intracellular concentration of another molecule (the xe2x80x9csecond messengerxe2x80x9d); calcium is known to play the role of first or second messenger in numerous cellular signaling contexts.
Calcium homeostasis in blood and other extracellular fluids is tightly controlled through the actions of calciotropic hormones on bone, kidneys, and intestine. In humans, dietary intake of calcium approximates 500 to 1000 mg/day, and obligatory endogenous losses in stool and urine total about 250 mg/day. On the order of 30% of calcium in the diet must be absorbed to sustain bone growth in children and to prevent postmenopausal bone loss in aging women. To meet the body""s need for calcium, the intestines of most vertebrates evolved specialized vitamin D-dependent and -independent mechanisms for ensuring adequate intestinal calcium uptake. Intestinal absorption of Ca2+ occurs by through both a saturable, transcellular process and a nonsaturable, paracellular pathway. When dietary calcium is abundant, the passive paracellular pathway is thought to be predominant. In contrast, when dietary calcium is limited, the active, vitamin D-dependent transcellular pathway plays a major role in calcium absorption.
The transcellular intestinal-uptake pathway is a multistep process, consisting of entry of luminal Ca2+ into an intestinal epithelial cells (i.e., an enterocyte), translocation of Ca2+ from its point of entry (the microvillus border of the apical plasma membrane) to the basolateral membrane, followed by active extrusion from the cell. Intracellular Ca2+ diffusion is thought to be facilitated by a calcium binding protein, calbindin D9K, whose biosynthesis is dependent on vitamin D. The extrusion of Ca2+ takes place against an electrochemical gradient and is mainly mediated by Ca-ATPase. The entry of Ca2+ across the apical membrane of the enterocyte is strongly favored electrochemically because the concentration of Ca2+ within the cell (10xe2x88x927-10xe2x88x926 M) is considerably lower than that in the intestinal lumen (10xe2x88x923 M) and the cell is electronegative relative to the intestinal lumen; as a result, the movement of Ca2+ across the apical membrane does not require the expenditure of energy.
The molecular mechanism responsible for entry of Ca2+ into intestinal cells has, however, been difficult to characterize. In particular, researchers have disagreed as to whether a transporter or a channel is responsible for this process (although studies have indicated that Ca2+ entry is voltage-independent and largely insensitive to classic L-type calcium channel blockers).
Brief Summary of the Invention
The present invention is directed, in a first aspect, toward a membrane protein that functions to transport calcium across cellular membranes. Our data indicate that this protein plays a key role in mediating Ca2+ entry into enterocytes as the first step of transcellular intestinal calcium absorption. Expression of the human homologue can be detected in placenta, pancreas, prostate, kidney, the gastrointestinal tract (e.g., esophagus, stomach, duodenum, jejunum, colon), liver, hair follicles, and testis, and is also expressed in cancer cell lines (specifically, chronic myelogenous leukemia cell line K-562 and colorectal adenocarcinoma cell line SW480). The rat isoform is expressed in rat intestine (although not in rat kidney). Thus, in contrast to the rat isoform, the human protein may be involved in the absorption/resorption of calcium in both intestine and kidney. Dysfunction of the human protein may be implicated in hyper- and hypocalcemia and calciuria, as well as in bone diseases, leukemia, and cancers affecting the prostate, breast, esophogus, stomach, and colon.
One embodiment of the invention comprises, as a composition of matter, a non-naturally occurring calcium-transport protein. Preferably, the transporter is a polypeptide encoded by a nucleic acid sequence within Seq. I.D. No. 1 or 3. In this context, the term xe2x80x9cencodedxe2x80x9d refers to an amino-acid sequence whose order is derived from the sequence of the nucleic acid or its complement. The nucleic acid sequence represented by Seq. I.D. No. 1 is derived from human sources. The nucleic acid sequence represented by Seq. I.D. No. 3 is derived from rat.
One aspect of this embodiment is directed toward a transporter having an amino-acid sequence substantially corresponding at least to the conserved regions of Seq. I.D. Nos. 2 or 4. The term xe2x80x9csubstantially,xe2x80x9d in this context, refers to a polypeptide that may comprise substitutions and modifications that do not alter the physiological activity of the protein to transport calcium across cellular membranes. The polypeptide represented by Seq. I.D. No. 2 is derived from human sources. The peptide represented by Seq. I.D. No. 4 is derived from rat.
In a second aspect, the invention pertains to a non-naturally occurring nucleic acid sequence encoding a calcium-transport protein. One embodiment of this aspect of the invention is directed toward a transporter having a nucleotide sequence substantially corresponding at least to the conserved regions of Seq. I.D. Nos. 2 or 4. The term xe2x80x9csubstantially,xe2x80x9d in this context, refers to a nucleic acid that may comprise substitutions and modifications that do not alter encoding of the amino-acid sequence, or which encodes a polypeptide having the same physiological activity in transporting calcium across cellular membranes. The term xe2x80x9ccorrespondingxe2x80x9d means homologous or complementary to a particular nucleic-acid sequence.
As used herein, the term xe2x80x9cnon-naturally occurring,xe2x80x9d in reference to a cell, refers to a cell that has a non-naturally occurring nucleic acid or a non-naturally occurring polypeptide, or is fused to a cell to which it is not fused in nature. The term xe2x80x9cnon-naturally occurring nucleic acidxe2x80x9d refers to a portion of genomic nucleic acid, a nucleic acid derived (e.g., by transcription) thereof, cDNA, or a synthetic or semi-synthetic nucleic acid which, by virtue of its origin or isolation or manipulation or purity, is not present in nature, or is linked to another nucleic acid or other chemical agent other than that to which it is linked in nature. The term xe2x80x9cnon-naturally occurring polypeptidexe2x80x9d or xe2x80x9cnon-naturally occurring proteinxe2x80x9d refers to a polypeptide which, by virtue of its amino-acid sequence or isolation or origin (e.g., synthetic or semi-synthetic) or manipulation or purity, is not present in nature, or is a portion of a larger naturally occurring polypeptide, or is linked to peptides, functional groups or chemical agents other than that to which it is linked in nature.
A third aspect of the present invention comprises a method of transporting calcium across a cellular membrane having a calcium transporter in accordance herewith. Calcium (in the divalent ionic form) is applied to the cellular membrane under conditions that allow the transporter to transport the calcium.
The cellular membrane can be derived, for example, from placenta, pancreas, prostate, kidney, the gastrointestinal tract (e.g., esophagus, stomach, duodenum, jejunum, colon), liver, or testis; or may be one of these tissues either in vivo or ex vivo. In practicing the method, the cell(s) giving rise to the cellular membrane may be transformed with the nucleic acid of Seq. I.D. Nos. 1 or 3 and maintained under conditions favoring functional expression of the transporter. A cell may be monitored for expression of the transporter by measuring the presence of calcium in the cell or transmembrane current flow. The invention also extends to a cell so transformed (e.g., a Xenopus laevis oocyte as described below).
In a fourth aspect, the invention comprises a method of identifying chemicals capable of interacting with the transporter, whether the protein is integral with a cellular membrane or present as a free species. Such chemicals may include antibodies or other targeting molecules that bind to the protein for purposes of identification, or which affect (e.g., by modulation or inhibition) the transport properties of the protein; and transportable species other than calcium.
In a fifth aspect, the invention comprises a method of blocking or inhibiting the uptake of calcium by cells having a calcium-transport protein in accordance herewith. In one embodiment, the method comprises the steps of causing an antibody or other targeting molecule to bind to the protein in a manner that inhibits calcium transport. In another embodiment, a nucleic acid complementary to at least a portion of the nucleic acid encoding the calcium-transport protein is introduced into the cells. The complementary nucleic acid blocks functional expression of the calcium-transport protein.